The goal of this research is to establish a general mechanism of ion channel gating for potassium channels in pursuit of a functional model for use in therapeutic development. The ATP-sensitive potassium (KATP) channel regulates ion flow across the plasma membrane based on interactions with relative levels of intracellular nucleotides. It is a principal player in many organ systems for controlling cell excitability;genetic mutations identified in patients have correlated channel dysfunction with disease;and several channel ligands have been developed to treat KATP-related disorders. Similar to other K+ channels and ion channels in general, KATP channels and other members of the inward rectifier (Kir) channel family have an intrinsic mechanism for restricting ion passage through changes in their conformation. The stochastic opening or closing of ion channels - termed "gating" - and the conformational changes in the protein complex that lead to gating are fundamental to understanding their role in physiology. This work will examine which structural elements of the KATP channel in the pore are involved in gating by utilizing known disease-causing mutations and selective mutagenesis to probe function with electrophysiology. Current structural and functional analysis of Kir channels suggest the M2 helices of Kir6.2, which line the pore of the channel, bend at a glycine position (the hinge) and splay apart at the intracellular end to open the pore at the helix bundle crossing. A preliminary examination of the upper glycine (G156 near selectivity filter) in the M2 helices indicates a necessary structural role for this residue during gating. Aim 1 characterized a clinical mutation G156R that produced complete loss of channel activity at the surface but was rescued with a second pore mutation, and further experiments elucidate implications of reconstituting gating function. The next two Aims examine the two transmembrane gates - selectivity filter (Aim 2) and helix bundle crossing (Aim 3) - and their dependence on the G156 residue to determine whether each domain works individually or cooperatively to gate the pore. The Aims will also enable me to draw conclusions about the validity of the hinge hypothesis in KATP channels. ATP-sensitive potassium channels selectively conduct potassium ions through changes in their structural conformation. Understanding what elements of protein structure are important for conformational transitions between one functional state and another will enable drug design to target a specific state of the channel to control its function. Selective drugs will improve alleviation of many disorders caused or ameliorated by the KATP channel.